ANALYSIS OF FAT-SOLUBLE VITAMINS USING UPLC-PDA-SQD Antonietta Gledhill Waters Corporation, Manchester, UK SUMMARY A 5-minute reversed phase analysis of fat-soluble vitamins is described for the simultaneous detection of vitamins A, D, E and K using the Waters ® ACQUITY UPLC ® System with the PDA and SQ Detector. The ACQUITY UPLC System is an advanced separation system that utilizes a 1.7 μm stationary phase particle size to improve resolution and peak shape in a shorter runtime. The SQ Detector is compatible with both Waters Empower™ and MassLynx™ Software. The MS setup parameters are made easy with the functionality of IntelliStart™ Technology, which has been incorporated into both software packages. INTRODUCTION Vitamins are essential nutrients that perform various roles to main- tain good health. Eating a normal, well-balanced diet should provide sufficient levels of vitamins; however, modern-day life-styles can lead to deficiencies. As a result, the practice of enriching foods with vitamins in order to provide the recommended daily allowance (RDA) has become commonplace. Vitamin A (retinol) plays an important role in bone growth, tooth development, reproduction, cell division, and gene expression. Sources of vitamin A are dairy products, carrots, pumpkin, dark green leafy vegetables, and apricots. Vitamin D helps increase the amounts of calcium absorbed from the small intestine and helps form and maintain bones. Children need adequate amounts of vitamin D to help develop strong bones and healthy teeth. Common sources of vitamin D include a variety enriched dairy products, most notably milk. Vitamin D can also be obtained through sunlight exposure of the skin. Vitamin E is a fat-soluble anti-oxidant, and protects vitamins A and C, red blood cells and essential fatty acids from destruction. Sources of vitamin E are margarines and vegetable oils. Vitamin K plays an essential role in normal blood clotting and helps promote bone health. Sources of vitamin K are green vegetables (spinach, broccoli), cauliflower, cabbage, and certain vegetable oils (soybean oil, cottonseed oil, canola oil, and olive oil). EXPERIMENTAL UPLC ® conditions LC system: ACQUITY UPLC Solvent A: Water:acetonitrile (90:10) Solvent B: Acetonitrile:methanol (50:50) Flow rate: 0.7 mL/min Column temp: 35 °C Column: ACQUITY ® BEH C 18 2.1 mm x 100 mm, 1.7 μm ACQUITY UPLC with PDA and SQ Detector ACQUITY UPLC with PDA and SQ Detector. G raphic box Use Futura Book 9 pt when labeling graphics B ox graphic with ¾ pt 50% black line G raphic box Use Futura Book 9 pt when labeling graphics B ox graphic with ¾ pt 50% black line
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ANALYSIS OF FAT-SOLUBLE VITAMINS USING UPLC-PDA-SQD · 2.25 Vitamin K 2 265 1.20-2.70 445 245 2.37 2Vitamin D 2 265 2.10-2.60 397 2.43 Vitamin D 3 265 2 2.20-2.80 385 2.77 Vitamin
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A NA LYSIS O F FAT-SO LU B L E V I TAM INS US ING U P L C- P DA-S Q D
Antonietta Gledhill Waters Corporation, Manchester, UK
SummaryA 5-minute reversed phase analysis of fat-soluble vitamins is described
for the simultaneous detection of vitamins A, D, E and K using the
Waters® ACQUITY UPLC® System with the PDA and SQ Detector.
The ACQUITY UPLC System is an advanced separation system that
utilizes a 1.7 μm stationary phase particle size to improve resolution
and peak shape in a shorter runtime.
The SQ Detector is compatible with both Waters Empower™ and
MassLynx™ Software. The MS setup parameters are made easy
with the functionality of IntelliStart™ Technology, which has been
incorporated into both software packages.
INT rODuCT IONVitamins are essential nutrients that perform various roles to main-
tain good health. Eating a normal, well-balanced diet should provide
sufficient levels of vitamins; however, modern-day life-styles can
lead to deficiencies. As a result, the practice of enriching foods
with vitamins in order to provide the recommended daily allowance
(RDA) has become commonplace.
Vitamin A (retinol) plays an important role in bone growth, tooth
development, reproduction, cell division, and gene expression.
Sources of vitamin A are dairy products, carrots, pumpkin, dark
green leafy vegetables, and apricots.
Vitamin D helps increase the amounts of calcium absorbed from
the small intestine and helps form and maintain bones. Children
need adequate amounts of vitamin D to help develop strong bones
and healthy teeth. Common sources of vitamin D include a variety
enriched dairy products, most notably milk. Vitamin D can also be
obtained through sunlight exposure of the skin.
Vitamin E is a fat-soluble anti-oxidant, and protects vitamins A
and C, red blood cells and essential fatty acids from destruction.
Sources of vitamin E are margarines and vegetable oils.
Vitamin K plays an essential role in normal blood clotting and helps
promote bone health. Sources of vitamin K are green vegetables
(spinach, broccoli), cauliflower, cabbage, and certain vegetable oils
(soybean oil, cottonseed oil, canola oil, and olive oil).
EX PErImENTaL
uPLC® conditionsLC system: ACQUITY UPLC
Solvent A: Water:acetonitrile (90:10)
Solvent B: Acetonitrile:methanol (50:50)
Flow rate: 0.7 mL/min
Column temp: 35 °C
Column: ACQUITY® BEH C18
2.1 mm x 100 mm, 1.7 μm
ACQUITY UPLC with PDA and SQ Detector
ACQUITY UPLC with PDA and SQ Detector.
G raphic boxUs e F utura B ook 9 pt when labeling graphics
B ox graphic with ¾ pt 50% black line
G raphic boxUs e F utura B ook 9 pt when labeling graphics
SQ Detector settingsThe single quadrupole MS was run in APCI mode.
Corona current: 15.0 uA
Cone voltage: 35.0 V
Source temp: 145 °C
Probe temp: 575 °C
Cone gas: 200 L/Hr
Desolvation gas: 600 L/Hr
(See Table 2 for the m/z values used for the SIR method)
Data acquisition and processingThe total system was controlled with MassLynx Software (creation
of methods and data-processing). The SQ Detector parameters were
set up via the IntelliStart Software and using the on-line fluidics.
IntelliStart monitors the performance of the mass spectrometer
and will initiate corrective action if any problems are diagnosed.
Processes such as tuning and calibration can be performed by the
click of a button using the on-line fluidics.
rESuLTS aND DISCuSSIONPublished methods for the detection of fat-soluble vitamins can
require 15 to 60 minutes. A standard solution of vitamins is shown
in Figure 1 with an analysis time of 5 minutes by UPLC. Table 2
shows the values used for the each vitamins analyzed.
RT Vitamin UVmax SIR - RT SIR -m/z windows
0.98 Vitamin A1 325 0.00-1.40 269
2.25 Vitamin K2
265 1.20-2.70 445
245
2.37 Vitamin D2 265 2 2.10-2.60 397
2.43 Vitamin D3 265 2 2.20-2.80 385
2.77 Vitamin E 291 2.60-3.10 431
3.02 Vitamin E acetate 285 2.80-3.40 473
3.24 Vitamin K1
265 3.10-3.60 451
240
Table 2. Retention times, UV maxima and m/z values (used for SIR) for the fat-soluble vitamins.
Vitamin A is the first to elute and Vitamin K is the last to elute on
the ACQUITY BEH C18 Column.
Figure 1. Standard mixture of fat-soluble vitamins.
Figure 3. PDA chromatogram of butter using the extended method where the presence of vitamin E (maxima 291) and vitamin E acetate (maxima 285) have been confirmed by their UV spectra (shown at the right hand side of the figure).
Figure 2 shows the presence of vitamin E (m/z 431) and
vitamin E acetate (m/z 473) from the standard mixture (left) and
from a butter sample (right) that has undergone liquid-liquid
extraction.1 Selected ion recording (SIR) was used to increase the
selectivity of the vitamins in the butter.
Figure 3 shows the resulting data from PDA detection. While the
chromatogram can be simplified for this particular butter sample by
selecting the specific maximas for each vitamin, the complexity of
certain systems can cause interferences that prevent comprehensive
analysis via a single detector. By employing complementary
techniques such as those described in this note it may be possible
to discern additional components and obtain required data.
Vitamin E acetateVitamin E acetate
Butter-Flora
Time
2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
6: SIR of 1 Channel AP+
473
5.96e7
5: SIR of 1 Channel AP+
431
2.41e7
Vitamin EVitamin E
Vit mix C18 column
Time
2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
1
2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
6: SIR of 1 Channel AP+
473
2.62e7
5: SIR of 1 Channel AP+
431
3.76e6
Figure 2. Vitamin E and Vitamin E acetate in standard solution (left), and in butter (right).
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, ACQUITY, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Empower, MassLynx, and IntelliStart, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.